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. 2011 May 4;6(5):e19457. doi: 10.1371/journal.pone.0019457

Figure 1. Approach for deciüphering the hairpin like structure of human bocavirus.

Figure 1

a: Putative hairpin structures of human bocavirus. In order to test for the self priming capability of the HBoV genome during genome replication different polymerases were added to HBoV DNA isolated from clinical samples. Both, positive strand and negative strand containing isolates were used [23]. Following self-priming elongation the DNA was denaturized and PCR was performed with a single primer. Surprisingly in none of the tested isolates a PCR product was observed, leading to the conclusion that no self priming occurred. b: HBoV end terminus PCR with primer Nrul1 and Ssp1, respectively. HBoV genome preparations were incubated with T7 polymerase and subject to subsequent PCR with primers Boca_end_NruI-1_neu and Boca_end_SspI-1_neu, respectively. Theoretically, elongation of terminal hairpin structures (self-priming) should have occurred as postulated in figure 1a. c: HBoV end terminus PCR with primer Nrul2 and Ssp2, respectively. HBoV genome preparations were incubated with Klenow polymerase and subject to subsequent PCR with primers NruI-2 and SspI-2, respectively. Theoretically, elongation of terminal hairpin structures (self-priming) should have occurred as postulated in figure 1a. d: HBoV end terminus PCR after circularisation of the genome with primer Nrul2 and Ssp2. HBoV genome preparations were incubated with T4 RNA ligase (self-ligation of single strand genomes and subject to subsequent PCR with primers Nru2 and Ssp2, respectively. Theoretically, head-to-tail sequences should have been amplified provided the terminal regions of the genome allow self-ligation and are not masked by secondary structures resistant to self-ligation. e: HBoV PCR with primer set Boca_end_SspI-1_neu and Boca_end_NruI-1_neu. This PCR approach was performed with primer in the outmost terminal region of the HBoV genome DQ000496. The PCR reaction should have amplified head-to-tail, tail-to-tail, or head-to-head structures, provided the target sequence is present in the clinical isolates. Unfortunately the primers did not bind to a terminal region that is know among all so far published isolates, thus it remains unclear whether primer binding was sufficient.